Transforming growth factor (TGF)-β is a multifunctional, homodimeric cytokine with a molecular weight of 25 kD, and exhibits a variety of biological activities.—e.g. It strongly promotes pathogenesis of sclerotic diseases including hepatic fibrosis/cirrhosis, atherosclerosis, lung fibrosis, scleroderma, renal failure (glomerulonephritis) or myelofibrosis as well as rheumatoid arthritis and proliferative vitreoretinopathy via stimulating the excessive production of extracellular matrices from mesenchymal cells and suppressing the growth of epithelial cells. Furthermore, TGF-β not only suppresses the growth and functions of skin keratinocytes, thereby causing their apoptosis and being deeply involved in epilation, but also suppresses functions of immune cells. It has been shown from the results of studies utilizing neutralizing antibodies against TGF-β in animal models that sclerotic diseases can be prevented or cured by suppressing the activities of TGF-β. For example, it has been reported to block TGF-β's activity at the entrance by employing either antibody therapies with neutralizing antibodies against TGF-β and its receptors, or gene therapies with dominant negative TGF-β receptor genes or soluble TGF-β receptor genes (JP Patent Publication (Kokai) No. 2004-121001 A; Schuppan et al., Digestion 59: 385-390, 1998; Qi et al., Proc Natl Acad Sci USA 96: 2345-2349, 1999; Ueno et al., Hum Gene Ther 11: 33-42, 2000). However since such an antibody therapy and a gene therapy retain problems in having an uncertainty in a necessary amount and an administration method, these methods cannot be immediately applied to the clinics. Thus, a challenge has been underway to develop a low molecular weight inhibitor based on the molecular mechanism of TGF-β's actions.
Using animal models, the present inventors demonstrated that a low molecular weight compound, cytoxazone, inhibits the signal transduction pathway of TGF-β, so as to suppress the pathogenesis of liver diseases (International Publication WO2005/039570). However, a target protein, on which cytoxazone directly acts, has not yet been identified.
On the other hand, by focusing on the fact that TGF-β is generated as an inactive latent molecule and converted to its active form by the action of protease (TGF-β activation reaction), the present inventors demonstrated a possibility that a low molecular weight synthetic protease inhibitor (Okuno et al., Gastroenterology 120: 18784-1800, 2001) as well as an antibody against the proteases (JP Patent Publication (Kokai) No. 2003-252792 A; Akita et al., Gastroenterology 123: 352-364, 2002) can be used to inhibit the TGF-β activation reaction, thereby preventing the diseases in animal models. However, since the current protease inhibitors have a wide range of inhibitory spectrum, there is a severe concern of side effects when applied to humans. Furthermore, since relatively high concentrations will be required for the current antibodies to work, there is a big concern of high costs due to a requirement for purification of a large amount of antibodies when applied to humans.
Moreover, the present inventors identified the cleavage site during the aforementioned TGF-β activation reaction, and succeeded in producing antibodies that recognize the cutting ends, and thus in demonstrating for the first time in the world that the proteolytic TGF-β activation reaction plays an important role in the pathogenesis of human liver diseases using these antibodies (International Publication WO2005/023870).
In the past, a peptide consisting of 8 to 17 amino acids that suppresses the generation of TGF-β in an immortalized hair papilla cell line was reported (JP Patent Publication (Kokai) No. 2005-239695 A). However, underlying action mechanisms thereof, including the possibilities of suppressing gene expression and/or activation reaction, as well as the effectiveness thereof in vivo, has not been shown.
Moreover, the group of Murphy-Ullrich at the University of Alabama, U.S.A. investigated the adherent-type TGF-β activation reaction by a glycoprotein, thrombospondin 1, and reported that TGF-β is activated by binding of KRFK sequence corresponding to amino acid numbers 412-415 of thrombospondin 1 to LSKL sequence corresponding to amino acid numbers 54-57 of LAP, and that the synthetic KRFK peptide activates TGF-β, whereas the synthetic LSKL peptide inhibits the TGF-β activation reaction (Schultz-Cherry et al., J. Biol. Chem. 270: 7304-7310, 1995; Crawford et al., Cell 93: 1159-1170, 1998; Ribeiro et al., J. Biol. Chem. 274: 13586-13593, 1999; Murphy-Ullrich and Poczatek Cytokine & Growth Factor Reviews 11: 59-69, 2000). The LSKL sequence derived from amino acid numbers 54-57 of the aforementioned LAP overlaps with the plasmin cleavage site K56-L57 identified by the present inventors. By binding to and cutting this portion, respectively, thrombospondin 1 and proteases may inhibit a noncovalent bond between this portion and active TGF-β molecule, resulting in release of the active TGF-β molecule from a latent complex (namely the TGF-β activation reaction). The present inventors, however, could not reproduce both the induction of TGF-β activation by the synthetic KRFK peptide and inhibition of TGF-β activation by the synthetic LSKL peptide, in their additional tests. Murphy-Ullrich et al. indicated an involvement of a thrombospondin 1-dependent adherent-type TGF-β activation reaction in the pathogenesis of lung or pancreatic diseases, but not in the pathogenesis of liver diseases.